This invention relates to a novel microfluidic device and methods of using this device to conduct in vitro studies on the reaction and effects of various compounds on cells.
The following references are cited in the application.
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The in vitro manipulation, study and processing of individual cells continues to be of importance both for theoretical evaluation as well as for the in vitro assaying of compounds for biological activity in such cells. However, conventional biological assay systems such as flow cytometry and cell perfusion chambers are typically operated with from 1 ml to 100 ml of reagents or more. A further disadvantage of techniques involving large volumes of cells is the inability to observe the effects on a cell both before, during and after it comes into contact with a candidate compound. Finally, statistical variations within a population of cells can limit the ability to resolve the effect of a compound.
Recently small disposable devices have been developed for handling biological samples and for conducting in vitro experiments on a controlled basis. For example, microchips have been used to electrophoretically separate mixtures of amino acids (1). Fluri et al. (2) also describe an integrated capillary electrophoresis device where electrophoresis is used to separate mixtures of amino acids.
The manipulation of a single cell by its electrophoretic mobility has been shown in a capillary. (3). Microchips have been designed to evaluate sperm function, principally motility, for in vitro fertilization. (4).
Analysis of the effects of candidate compounds on cell function demands careful handling of candidate compounds which are often limited in both quantity and concentration. The ability to observe the effect of the candidate compounds on individual cells in a device potentially suitable for a high level of multiplexing makes miniaturized analysis very attractive. Furthermore, the ability to observe the effect of a candidate compound on non-adherent cells would be of benefit.
Microfluidic systems embodied in a microchip would use small volumes, providing cost saving advantages for work involving expensive reagents, especially candidate compounds made for new drug screening and of course would reduce the amount of candidate compound required.
The ability to sort cell responses into classes and analyze each class separately would reduce the apparent statistical variation seen when large number of cells are evaluated en masse. Single cell studies would also allow the progression of events within a single cell to be evaluated, in contrast to flow cytometry where a progression of events is studied over an ensemble of cells. Statistical variations within an ensemble can limit the ability to resolve a particular effect, whereas working with individual cells will maximize resolution and signal to noise for a given event.
It would be therefore advantageous to manipulate and transport cells within a microfabricated reaction device thereby allowing the observation of the cell reactions.
The present invention is directed to a method of observing the effect of a compound or a mixture of compounds on cells in a microfluidic device having a main flow path having a detection zone, at least two inlet flow paths intersecting and merging with the main flow path at or upstream of the detection zone; applying at least one cell to a first inlet flow path and the desired compound to a second inlet flow path; inducing flow of the cells and the desired compound toward the outlet; allowing the cells to mix with the desired compound at the intersection of the second inlet flow path and the main flow path; and observing the effect of the compound on the cells in the detection zone.
In one of its method aspects the present invention is directed to a method for studying calcium influx on a cell in a microfluidic system having a main flow path having a detection zone and an outlet, at least two inlet flow paths sequentially intersecting and merging with the main flow path upstream of the detection zone applying lymphocytes to a first inlet flow path and an activator to a second inlet flow path; inducing flow of the lymphocytes and the activator toward the outlet; allowing the lymphocytes to interact with the activator at the intersection of the second inlet and the main flow path; and observing the effect of the activator on the lymphocytes in the detection zone. The method wherein the device further comprises a third inlet flow path which intersects with the main flow path upstream of the detection zone and an inhibitor is added to the third inlet flow path and the effect of the inhibitor is observed in the detection zone.
In another of its method aspects the present invention is directed to a method for studying leukocyte rolling comprising a microfluidic system having a main flow path with a detection zone and an outlet, at least two inlet flow paths intersecting into the main flow path upstream of the detection zone and wherein the walls of main flow path in the detection zone have attached thereto a cell adhesion molecule; applying at least one leukocyte cell to a first inlet flow path; applying a candidate compound to a second inlet flow path; inducing flow of the cells and the candidate compound toward the outlet; allowing the leukocytes; candidate compound and the cell adhesion molecules to interact; and observing the leukocyte rolling in the detection zone. The method wherein the device further comprises at least two detection zones wherein the walls of the main flow path in the first detection zone are free of adhesion molecules and the walls of the main flow path in the second detection zone have adhesion molecules attached thereto and observing the rolling of the leukocytes in both detection zones.
In one of its product aspects the present invention is directed to a microfluidic device comprising a main flow path comprising a detection zone and an outlet; at least two inlet flow paths intersecting in fluid communication with the main flow path at or upstream of the detection zone at an upstream angle of less than 90xc2x0.
In another of its product aspects the present invention is directed to an observation device comprising a plurality of the microfluidic devices of the present invention wherein the main flow paths of the microfluidic devices are substantially parallel at their detection zones.